Justine Meiller

The interaction of bortezomib with multidrug transporters: implications for therapeutic applications in advanced multiple myeloma and other neoplasias

PurposeBortezomib is an important agent in multiple myeloma treatment, but resistance in cell lines and patients has been described. The main mechanisms of resistance described in cancer fall into one of two categories, pharmacokinetic resistance (PK), e.g. over expression of drug efflux pumps and pharmacodynamic resistance, e.g. apoptosis resistance or altered survival pathways, where the agent reaches an appropriate concentration, but this fails to propagate an appropriate cell death response. Of the known pump mechanisms, P-glycoprotein (P-gp) is the best studied and considered to be the most important in contributing to general PK drug resistance. Resistance to bortezomib is multifactorial and there are conflicting indications that cellular overexpression of P-gp may contribute to resistance agent. Hence, better characterization of the interactions of this drug with classical resistance mechanisms should identify improved treatment applications.MethodsCell lines with different P-gp expression levels were used to determine the relationship between bortezomib and P-gp. Coculture system with stromal cells was used to determine the effect of the local microenvironment on the bortezomib–elacridar combination. To further assess P-gp function, intracellular accumulation of P-gp probe rhodamine-123 was utilised.ResultsIn the present study, we show that bortezomib is a substrate for P-gp, but not for the other drug efflux transporters. Bortezomib activity is affected by P-gp expression and conversely, the expression of P-gp affect bortezomib’s ability to act as a P-gp substrate. The local microenvironment did not alter the cellular response to bortezomib. We also demonstrate that bortezomib directly affects the expression and function of P-gp.ConclusionsOur findings strongly support a role for P-gp in bortezomib resistance and, therefore, suggest that combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable treatment combination to extend efficacy of this important drug.

A novel panel of protein biomarkers for predicting response to thalidomide-based therapy in newly diagnosed multiple myeloma patients

Multiple myeloma (MM) is a heterogeneous group of disorders both genotypically and phenotypically. Response to thalidomide‐based induction therapy in newly diagnosed patients varies significantly in published clinical trials. Proteomic analysis was performed on 39 newly diagnosed MM patients treated with a thalidomide‐based regimen (22 responders; 17 non‐responders) using immunodepletion, 2‐D DIGE analysis and mass spectrometry. Zinc‐α‐2‐glycoprotein (ZAG), vitamin D‐binding protein (VDB), serum amyloid‐A protein (SAA) and β‐2‐microglobulin (B2M) had statistically significant higher concentrations in non‐responders compared to responders, while haptoglobin (Hp) had a lower concentration. ELISAs were used to validate the candidate protein biomarkers using unfractionated serum from 51 newly diagnosed MM patients (29 responders; 22 non‐responders). Using logistic regression, the best possible area under the curve (AUC) was 0.96 using ZAG, VDB and SAA in combination. Leave‐one‐out‐cross‐validation (LOOCV) indicated an overall predictive accuracy of 84% with associated sensitivity and specificity values of 81.8 and 86.2%, respectively. Subsequently, 16 of 22 thalidomide‐refractory patients successfully achieved complete response or very good partial response using second‐line treatment suggesting that the biomarker profile is specific to thalidomide response rather than identifying patients with MM refractory to all therapies. Using a novel panel of predictive biomarkers, the feasibility of predicting response to thalidomide‐based therapy in previously untreated MM has been demonstrated.

Elevated levels of 14-3-3 proteins, serotonin, gamma enolase and pyruvate kinase identified in clinical samples from patients diagnosed with colorectal cancer

BACKGROUND Colorectal cancer (CRC), a heterogeneous disease that is common in both men and women, continues to be one of the predominant cancers worldwide. Lifestyle, diet, environmental factors and gene defects all contribute towards CRC development risk. Therefore, the identification of novel biomarkers to aid in the management of CRC is crucial. The aim of the present study was to identify candidate biomarkers for CRC, and to develop a better understanding of their role in tumourogenesis. METHODS In this study, both plasma and tissue samples from patients diagnosed with CRC, together with non-malignant and normal controls were examined using mass spectrometry based proteomics and metabolomics approaches. RESULTS It was established that the level of several biomolecules, including serotonin, gamma enolase, pyruvate kinase and members of the 14-3-3 family of proteins, showed statistically significant changes when comparing malignant versus non-malignant patient samples, with a distinct pattern emerging mirroring cancer cell energy production. CONCLUSION The diagnosis and management of CRC could be enhanced by the discovery and validation of new candidate biomarkers, as found in this study, aimed at facilitating early detection and/or patient stratification together with providing information on the complex behaviour of cancer cells.

Identification of proteins found to be significantly altered when comparing the serum proteome from Multiple Myeloma patients with varying degrees of bone disease

BackgroundBone destruction is a feature of multiple myeloma, characterised by osteolytic bone destruction due to increased osteoclast activity and suppressed or absent osteoblast activity. Almost all multiple myeloma patients develop osteolytic bone lesions associated with severe and debilitating bone pain, pathologic fractures, hypercalcemia, and spinal cord compression, as well as increased mortality. Biomarkers of bone remodelling are used to identify disease characteristics that can help select the optimal management of patients. However, more accurate biomarkers are needed to effectively mirror the dynamics of bone disease activity.ResultsA label-free mass spectrometry-based strategy was employed for discovery phase analysis of fractionated patient serum samples associated with no or high bone disease. A number of proteins were identified which were statistically significantly correlated with bone disease, including enzymes, extracellular matrix glycoproteins, and components of the complement system.ConclusionsEnzyme-linked immunosorbent assay of complement C4 and serum paraoxonase/arylesterase 1 indicated that these proteins were associated with high bone disease in a larger independent cohort of patient samples. These biomolecules may therefore be clinically useful in assessing the extent of bone disease.

Development, validation and application of a sensitive LC–MS/MS method for the quantification of thalidomide in human serum, cells and cell culture medium

A simple, robust, sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of thalidomide was developed and validated. The method was applied to thalidomide quantification in three different types of biological samples. Thalidomide was extracted from human serum (100 μL), cells (2.5 × 10(5)), or cell culture media (100 μL) by LLE and separated on a Prodigy C18 (150 mm × 4.0 mm, 5 μm i.d.) column with isocratic elution using water/acetonitrile (70/30, v/v) 0.1% formic acid, at a flow rate of 0.5 mL/min, with umbelliferone (600 ng/mL) as an internal standard. Thalidomide was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring mode using positive electrospray ionisation. The method was validated in two separate thalidomide concentration ranges; human serum (0.05-20 μg/mL) and in vitro cells (0.78-50 ng) with an inter-day precision of 1.8% and 1.9% and average accuracy of 100% and 101% in serum and cells respectively. Despite the use of small sample volume, the limit of quantification for thalidomide in serum was determined to be 3 ng/mL. The method was successfully employed to measure levels of thalidomide in cancer patient serum and cell culture model systems. Although cellular levels were quantifiable, thalidomide was shown to be unstable under in vitro conditions with a half life of approximately 2 h. In patient samples, circulating serum levels showed a broad correlation with dose and uncovered some patient compliance issues.

Clinical utility of C-terminal telopeptide of type 1 collagen in multiple myeloma.

Myeloma bone disease (MBD) is a major cause of morbidity in multiple myeloma (MM). We investigated bone turnover markers (BTM) as relapse predictors and biomarkers for monitoring MBD. We measured C‐terminal telopeptide of type I collagen (CTX‐1), and Procollagen type 1 N Propeptide (P1NP) in 86 MM patients and 26 controls. CTX‐1 was higher in newly diagnosed patients compared to control, remission and relapse (P < 0·05), and decreased following treatment. In the setting of relapse, a CTX‐1 rise greater than the calculated least significant change (LSC) was observed in 26% of patients 3–6 months prior to relapse (P = 0·007), and in 60·8% up to 3 months before relapse (P = 0·015). Statistically significant changes in CTX‐1 levels were also observed in patients who were with and without bisphosphonate therapy at the time of relapse. In patients with normal renal function, mean CTX‐1 level was highest in the newly diagnosed group (0·771 ± 0·400 μg/l), and lowest in the remission group (0·099 ± 0·070 μg/l) (P < 0·0001). P1NP levels were not statistically different across the patient groups. We conclude that CTX‐1, measured on an automated hospital laboratory platform, has a role in routine treatment monitoring and predicting relapse of MBD, even in patients on bisphosphonates.

Novel panel of protein biomarkers to predict response to bortezomib-containing induction regimens in multiple myeloma patients

Background Multiple myeloma (MM) is a complex heterogeneous disease. Various risk stratification models have been recommended including cytogenetic and FISH analysis to identify high-risk patients who may benefit from novel treatments, but such facilities are not widely available. The International Scoring System (ISS) using beta-2-microglobulin and albumin remains a widely used prognostic scoring system in many clinical practices; however it is not useful in predicting response to treatment in MM. The aim of this study is to identify clinically useful biomarkers to predict response to treatment containing bortezomib. Methods 17 MM patient serum samples (9 responders/8 non-responders) were used for the discovery phase (label-free mass spectrometry) and an additional 20 MM patient serum samples were used for the ELISA-based validation phase (14 responders/6 non-responders). Results CLU and ANG mean levels were higher in the responders group, while Complement C1q had lower concentrations. The combination of all standard biomarkers (albumin, beta-2-microglobulin (ß2M), paraprotein and kappa/lambda (K/L) ratio) had an AUC value of 0.71 with 65% correct classification, while an overall combination of new candidate protein biomarkers with standard biomarkers had an AUC value of 0.89 with 85.3% correct classification. Conclusions A combination of new and standard biomarkers consisting of CLU, ANG, C1Q, albumin, ß2M, paraprotein and K/L ratio may have potential as a novel panel of biomarkers to predict MM response to treatment containing bortezomib. General significance Use of this biomarker panel could facilitate a more personalized therapy approach and to minimize unnecessary side effects from ineffective drugs.

Ultra-deep next generation mitochondrial genome sequencing reveals widespread heteroplasmy in Chinese hamster ovary cells

Recent sequencing of the Chinese hamster ovary (CHO) cell and Chinese hamster genomes has dramatically advanced our ability to understand the biology of these mammalian cell factories. In this study, we focus on the powerhouse of the CHO cell, the mitochondrion. Utilizing a high-resolution next generation sequencing approach we sequenced the Chinese hamster mitochondrial genome for the first time and surveyed the mutational landscape of CHO cell mitochondrial DNA (mtDNA). Depths of coverage ranging from ~3,319X to 8,056X enabled accurate identification of low frequency mutations (>1%), revealing that mtDNA heteroplasmy is widespread in CHO cells. A total of 197 variants at 130 individual nucleotide positions were identified across a panel of 22 cell lines with 81% of variants occurring at an allele frequency of between 1% and 99%. 89% of the heteroplasmic mutations identified were cell line specific with the majority of shared heteroplasmic SNPs and INDELs detected in clones from 2 cell line development projects originating from the same host cell line. The frequency of common predicted loss of function mutations varied significantly amongst the clones indicating that heteroplasmic mtDNA variation could lead to a continuous range of phenotypes and play a role in cell to cell, production run to production run and indeed clone to clone variation in CHO cell metabolism. Experiments that integrate mtDNA sequencing with metabolic flux analysis and metabolomics have the potential to improve cell line selection and enhance CHO cell metabolic phenotypes for biopharmaceutical manufacturing through rational mitochondrial genome engineering.